This invention relates to protein scaffolds useful, for example, for the generation of products having novel binding characteristics.
Proteins having relatively defined three-dimensional structures, commonly referred to as protein scaffolds, may be used as reagents for the design of engineered products. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomization is often carried out to produce libraries of proteins from which desired products may be selected. One particular area in which such scaffolds are useful is the field of antibody design.
A number of previous approaches to the manipulation of the mammalian immune system to obtain reagents or drugs have been attempted. These have included injecting animals with antigens of interest to obtain mixtures of polyclonal antibodies reactive against specific antigens, production of monoclonal antibodies in hybridoma cell culture (Koehler and Milstein, Nature 256:495, 1975), modification of existing monoclonal antibodies to obtain new or optimized recognition properties, creation of novel antibody fragments with desirable binding characteristics, and randomization of single chain antibodies (created by connecting the variable regions of the heavy and light chains of antibody molecules with a flexible peptide linker) followed by selection for antigen binding by phage display (Clackson et al., Nature 352:624, 1991).
In addition, several non-immunoglobulin protein scaffolds have been proposed for obtaining proteins with novel binding properties. For example, a xe2x80x9cminibodyxe2x80x9d scaffold, which is related to the immunoglobulin fold, has been designed by deleting three beta strands from a heavy chain variable domain of a monoclonal antibody (Tramontano et al., J. Mol. Recognit. 7:9, 1994). This protein includes 61 residues and can be used to present two hypervariable loops. These two loops have been randomized and products selected for antigen binding, but thus far the framework appears to have somewhat limited utility due to solubility problems. Another framework used to display loops has been tendamistat, a 74 residue, six-strand beta sheet sandwich held together by two disulfide bonds (McConnell and Hoess, J. Mol. Biol. 250:460, 1995). This scaffold includes three loops, but, to date, only two of these loops have been examined for randomization potential.
Other proteins have been tested as frameworks and have been used to display randomized residues on alpha helical surfaces (Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Protein Eng. 8:601, 1995), loops between alpha helices in alpha helix bundles (Ku and Schultz, Proc. Natl. Acad. Sci. USA 92:6552, 1995), and loops constrained by disulfide bridges, such as those of the small protease inhibitors (Markland et al., Biochemistry 35:8045, 1996; Markland et al., Biochemistry 35:8058, 1996; Rottgen and Collins, Gene 164:243, 1995; Wang et al., J. Biol. Chem. 270:12250, 1995).
The present invention provides a new family of proteins capable of evolving to bind any compound of interest. These proteins, which make use of a fibronectin or fibronectin-like scaffold, function in a manner characteristic of natural or engineered antibodies (that is, polyclonal, monoclonal, or single-chain antibodies) and, in addition, possess structural advantages. Specifically, the structure of these antibody mimics has been designed for optimal folding, stability, and solubility, even under conditions which normally lead to the loss of structure and function in antibodies.
These antibody mimics may be utilized for the purpose of designing proteins which are capable of binding to virtually any compound (for example, any protein) of interest. In particular, the fibronectin-based molecules described herein may be used as scaffolds which are subjected to directed evolution designed to randomize one or more of the three fibronectin loops which are analogous to the complementarity-determining regions (CDRs) of an antibody variable region. Such a directed evolution approach results in the production of antibody-like molecules with high affinities for antigens of interest. In addition, the scaffolds described herein may be used to display defined exposed loops (for example, loops previously randomized and selected on the basis of antigen binding) in order to direct the evolution of molecules that bind to such introduced loops. A selection of this type may be carried out to identify recognition molecules for any individual CDR-like loop or, alternatively, for the recognition of two or all three CDR-like loops combined into a non-linear epitope.
Accordingly, the present invention features a protein that includes a fibronectin type III domain having at least one randomized loop, the protein being characterized by its ability to bind to a compound that is not bound by the corresponding naturally-occurring fibronectin.
In preferred embodiments, the fibronectin type III domain is a mammalian (for example, a human) fibronectin type III domain; and the protein includes the tenth module of the fibronectin type III (10Fn3) domain. In such proteins, compound binding is preferably mediated by either one, two, or three 10Fn3 loops. In other preferred embodiments, the second loop of 10Fn3 may be extended in length relative to the naturally-occurring module, or the 10Fn3 may lack an integrin-binding motif. In these molecules, the integrin-binding motif may be replaced by an amino acid sequence in which a basic amino acid-neutral amino acid-acidic amino acid sequence (in the N-terminal to C-terminal direction) replaces the integrin-binding motif; one preferred sequence is serine-glycine-glutamate. In another preferred embodiment, the fibronectin type III domain-containing proteins of the invention lack disulfide bonds.
Any of the fibronectin type III domain-containing proteins described herein may be formulated as part of a fusion protein (for example, a fusion protein which further includes an immunoglobulin Fc domain, a complement protein, a toxin protein, or an albumin protein). In addition, any of the fibronectin type III domain proteins may be covalently bound to a nucleic acid (for example, an RNA), and the nucleic acid may encode the protein. Moreover, the protein may be a multimer, or, particularly if it lacks an integrin-binding motif, it may be formulated in a physiologically-acceptable carrier.
The present invention also features proteins that include a fibronectin type III domain having at least one mutation in a xcex2-sheet sequence which changes the scaffold structure. Again, these proteins are characterized by their ability to bind to compounds that are not bound by the corresponding naturally-occurring fibronectin.
In addition, any of the fibronectin scaffolds of the invention may be immobilized on a solid support (for example, a bead or chip), and these scaffolds may be arranged in any configuration on the solid support, including an array.
In a related aspect, the invention further features nucleic acids encoding any of the proteins of the invention. In preferred embodiments, the nucleic acid is DNA or RNA.
In another related aspect, the invention also features a method for generating a protein which includes a fibronectin type III domain and which is pharmaceutically acceptable to a mammal, involving removing the integrin-binding domain of said fibronectin type III domain. This method may be applied to any of the fibronectin type III domain-containing proteins described above and is particularly useful for generating proteins for human therapeutic applications. The invention also features such fibronectin type III domain-containing proteins which lack integrin-binding domains.
In yet other related aspects, the invention features screening methods which may be used to obtain or evolve randomized fibronectin type III proteins capable of binding to compounds of interest, or to obtain or evolve compounds (for example, proteins) capable of binding to a particular protein containing a randomized fibronectin type III motif. In addition, the invention features screening procedures which combine these two methods, in any order, to obtain either compounds or proteins of interest.
In particular, the first screening method, useful for the isolation or identification of randomized proteins of interest, involves: (a) contacting the compound with a candidate protein, the candidate protein including a fibronectin type III domain having at least one randomized loop, the contacting being carried out under conditions that allow compound-protein complex formation; and (b) obtaining, from the complex, the protein which binds to the compound.
The second screening method, for isolating or identifying a compound which binds to a protein having a randomized fibronectin type III domain, involves: (a) contacting the protein with a candidate compound, the contacting being carried out under conditions that allow compound-protein complex formation; and (b) obtaining, from the complex, the compound which binds to the protein.
In preferred embodiments, the methods further involve either randomizing at least one loop of the fibronectin type III domain of the protein obtained in step (b) and repeating steps (a) and (b) using the further randomized protein, or modifying the compound obtained in step (b) and repeating steps (a) and (b) using the further modified compound. In addition, the compound is preferably a protein, and the fibronectin type III domain is preferably a mammalian (for example, a human) fibronectin type III domain. In other preferred embodiments, the protein includes the tenth module of the fibronectin type III domain (10Fn3), and binding is mediated by one, two, or three 10Fn3 loops. In addition, the second loop of 10Fn3 may be extended in length relative to the naturally-occurring module, or 10Fn3 may lack an integrin-binding motif. Again, as described above, the integrin-binding motif may be replaced by an amino acid sequence in which a basic amino acid-neutral amino acid-acidic amino acid sequence (in the N-terminal to C-terminal direction) replaces the integrin-binding motif; one preferred sequence is serine-glycine-glutamate.
The selection methods described herein may be carried out using any fibronectin type III domain-containing protein. For example, the fibronectin type III domain-containing protein may lack disulfide bonds, or may be formulated as part of a fusion protein (for example, a fusion protein which further includes an immunoglobulin Fc domain, a complement protein, a toxin protein, or an albumin protein). In addition, selections may be carried out using the fibronectin type III domain proteins covalently bound to nucleic acids (for example, RNAs or any nucleic acid which encodes the protein). Moreover, the selections may be carried out using fibronectin domain-containing protein multimers.
Preferably, the selections involve the immobilization of the binding target on a solid support. Preferred solid supports include columns (for example, affinity columns, such as agarose columns) or microchips.
In addition, the invention features diagnostic methods which employ the fibronectin scaffold proteins of the invention. Such diagnostic methods may be carried out on a sample (for example, a biological sample) to detect one analyte or to simultaneously detect many different analytes in the sample. The method may employ any of the scaffold molecules described herein. Preferably, the method involves (a) contacting the sample with a protein which binds to the compound analyte and which includes a fibronectin type III domain having at least one randomized loop, the contacting being carried out under conditions that allow compound-protein complex formation; and (b) detecting the complex, and therefore the compound in the sample.
In preferred embodiments, the protein is immobilized on a solid support (for example, a chip or bead) and may be immobilized as part of an array. The protein may be covalently bound to a nucleic acid, preferably, a nucleic acid, such as RNA, that encodes the protein. In addition, the compound is often a protein, but may also be any other analyte in a sample. Detection may be accomplished by any standard technique including, without limitation, radiography, fluorescence detection, mass spectroscopy, or surface plasmon resonance.
As used herein, by xe2x80x9cfibronectin type III domainxe2x80x9d is meant a domain having 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands. Preferably, a fibronectin type III domain includes a sequence which exhibits at least 30% amino acid identity, and preferably at least 50% amino acid identity, to the sequence encoding the structure of the 10Fn3 domain referred to as xe2x80x9c1ttgxe2x80x9d (ID=xe2x80x9c1ttgxe2x80x9d (one ttg)) available from the Protein Data Base. Sequence identity referred to in this definition is determined by the Homology program, available from Molecular Simulation (San Diego, Calif.). The invention further includes polymers of 10Fn3-related molecules, which are an extension of the use of the monomer structure, whether or not the subunits of the polyprotein are identical or different in sequence.
By xe2x80x9cnaturally occurring fibronectinxe2x80x9d is meant any fibronectin protein that is encoded by a living organism.
By xe2x80x9crandomizedxe2x80x9d is meant including one or more amino acid alterations relative to a template sequence.
By a xe2x80x9cproteinxe2x80x9d is meant any sequence of two or more amino acids, regardless of length, post-translation modification, or function. xe2x80x9cProteinxe2x80x9d and xe2x80x9cpeptidexe2x80x9d are used interchangeably herein.
By xe2x80x9cRNAxe2x80x9d is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. One example of a modified RNA included within this term is phosphorothioate RNA.
By xe2x80x9cDNAxe2x80x9d is meant a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides.
By a xe2x80x9cnucleic acidxe2x80x9d is meant any two or more covalently bonded nucleotides or nucleotide analogs or derivatives. As used herein, this term includes, without limitation, DNA, RNA, and PNA.
By xe2x80x9cpharmaceutically acceptablexe2x80x9d is meant a compound or protein that may be administered to an animal (for example, a mammal) without significant adverse medical consequences.
By xe2x80x9cphysiologically acceptable carrierxe2x80x9d is meant a carrier which does not have a significant detrimental impact on the treated host and which retains the therapeutic properties of the compound with which it is administered. One exemplary physiologically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington""s Pharmaceutical Sciences, (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa., incorporated herein by reference.
By xe2x80x9cselectingxe2x80x9d is meant substantially partitioning a molecule from other molecules in a population. As used herein, a xe2x80x9cselectingxe2x80x9d step provides at least a 2-fold, preferably, a 30-fold, more preferably, a 100-fold, and, most preferably, a 1000-fold enrichment of a desired molecule relative to undesired molecules in a population following the selection step. A selection step may be repeated any number of times, and different types of selection steps may be combined in a given approach.
By xe2x80x9cbinding partner,xe2x80x9d as used herein, is meant any molecule which has a specific, covalent or non-covalent affinity for a portion of a desired compound (for example, protein) of interest. Examples of binding partners include, without limitation, members of antigen/antibody pairs, protein/inhibitor pairs, receptor/ligand pairs (for example cell surface receptor/ligand pairs, such as hormone receptor/peptide hormone pairs), enzyme/substrate pairs (for example, kinase/substrate pairs), lectin/carbohydrate pairs, oligomeric or heterooligomeric protein aggregates, DNA binding protein/DNA binding site pairs, RNA/protein pairs, and nucleic acid duplexes, heteroduplexes, or ligated strands, as well as any molecule which is capable of forming one or more covalent or non-covalent bonds (for example, disulfide bonds) with any portion of another molecule (for example, a compound or protein).
By a xe2x80x9csolid supportxe2x80x9d is meant, without limitation, any column (or column material), bead, test tube, microtiter dish, solid particle (for example, agarose or sepharose), microchip (for example, silicon, silicon-glass, or gold chip), or membrane (for example, the membrane of a liposome or vesicle) to which a fibronectin scaffold or an affinity complex may be bound, either directly or indirectly (for example, through other binding partner intermediates such as other antibodies or Protein A), or in which a fibronectin scaffold or an affinity complex may be embedded (for example, through a receptor or channel).
The present invention provides a number of advantages. For example, as described in more detail below, the present antibody mimics exhibit improved biophysical properties, such as stability under reducing conditions and solubility at high concentrations. In addition, these molecules may be readily expressed and folded in prokaryotic systems, such as E. coli, in eukaryotic systems, such as yeast, and in vitro translation systems, such as the rabbit reticulocyte lysate system. Moreover, these molecules are extremely amenable to affinity maturation techniques involving multiple cycles of selection, including in vitro selection using RNA-protein fusion technology (Roberts and Szostak, Proc. Natl. Acad. Sci USA 94:12297, 1997; Szostak et al., U.S. Ser. No. 09/007,005, now U.S. Pat. No. 6,258,558 B1 and U.S. Ser. No. 09/247,190, now U.S. Pat. No. 6,261,804 B1; Szostak et al. WO98/31700), phage display (see, for example, Smith and Petrenko, Chem. Rev. 97:317, 1997), and yeast display systems (see, for example, Boder and Wittrup, Nature Biotech. 15:553, 1997).
Other features and advantages of the present invention will be apparent from the following detailed description thereof, and from the claims.